双向凝胶电泳中三种蛋白质检测方法的比较

高通量双向电泳是蛋白质组学的核心 ,双向电泳凝胶上蛋白质点的检测方法应具有灵敏度高、线性范围宽和兼容质谱鉴定等优点 .采用差异凝胶电泳 (differencegelelectrophoresis ,DIGE)技术以Cy3(1 (5 carboxypentyl) 1′ propylindocarbocyaninehalideN hydroxysuccinimidylester)和Cy5 (1 (5 carboxypentyl) 1′ methylindodicarbocyaninehalideN hydroxysuccinimidylester)荧光分别标记正常和TNF α处理细胞的蛋白质 ,用Cy2 (3 (4 carboxymethyl)phenylmethyl) 3′ ethyloxacarbocyaninehalideN hydroxysuccinimidylester)荧光标记正常和TNF α处理细胞蛋白质的等量混合样品作为内标 ,混合 3种荧光标记的蛋白质后 ,在同一等电聚焦胶条进行聚焦 ,然后在聚丙烯酰胺凝胶上进行第二向电泳 ,用 3种波长的激光激发扫描得到凝胶图象 ,DIGE中多个样品在同一条件下电泳 ,因而匹配率高 ,且引入内标使蛋白质点的检测与定量更为准确 .DIGE技术与质谱相结合 ,实现了高通量和相对准确定量 .与硝酸银和考马斯亮蓝染色结果相比较 ,DIGE技术具有灵敏度高、线性范围宽和不影响后续质谱鉴定等优点

Comparison of Three Protein Detect Methods in Two-Dimensional Gel Eletrophoresis

There is an increasing use of two dimensional gel electrophoresis (2D gel)in the proteomics.The 2 D gel is characterized with higher sensitivity,linear dynamic ranges and good reproducibility.This method is based on the principle:(1)Normal or TNF α treated protein is labeled with Cy3 (1 (5 carboxypentyl) 1′propyl indocarbocyanine halide N hydroxysuccinimidyl ester) and Cy5 (1 (5 carboxypentyl) 1′methyl indocarbocyanine halide N hydroxysuccinimidyl ester),and then the mixer of normal and TNF α treated cellular protein is labeled with Cy2(3 (4 carboxymethyl)phenylmethyl) 3′ etyloxacarbocyanine halide N hydroxysuccinimidyl ester)as an inner standard.(2) After these fluorescence labeled proteins are mixed together,samples are running on a same 2 D gel using a method referred to a different gel electrophoresis(DIGE).(3)Images of the 2 D gels are obtained using three different excitation filters.The ratio of the different colored fluorescent signals are used to distinguish the different proteins in the sample.Compared with traditional methods such as staining with AgNO 3 or Coomassie blue,the method DIGE has a higher matching rate and more accurate result.